title: Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis.
abstract: Posttranscriptional modifications of bacterial rRNA serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. Here, we investigated the functional role of one rRNA modification in particular-the methylation of guanosine at position 518 (G518) of the 16S rRNA in Mycobacterium tuberculosis. Based on previously reported evidence that G518 is located 5 Å; from proline 44 of ribosomal protein S12, which interacts directly with the mRNA wobble position of the codon:anticodon helix at the A site during translation, we speculated that methylation of G518 affects protein translation. We transformed reporter constructs designed to probe the effect of functional lesions at one of the three codon positions on translational fidelity into the wild-type strain, H37Rv, and into a ΔgidB mutant, which lacks the methyltransferase (GidB) that methylates G518. We show that mistranslation occurs less in the ΔgidB mutant only in the construct bearing a lesion in the wobble position compared to H37Rv. Thus, the methylation of G518 allows mistranslation to occur at some level in order for translation to proceed smoothly and efficiently. We also explored the role of methylation at G518 in altering the susceptibility of M. tuberculosis to streptomycin (SM). Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we confirmed that G518 is not methylated in the ΔgidB mutant. Furthermore, isothermal titration calorimetry experiments performed on 70S ribosomes purified from wild-type and ΔgidB mutant strains showed that methylation significantly enhances SM binding. These results provide a mechanistic explanation for the low-level, SM-resistant phenotype observed in M. tuberculosis strains that contain a gidB mutation.
authors.affiliation: Tuberculosis Research Section, Laboratory of Clinical Infectious Disease, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sharon Y
authors.initials: SY
authors.lastname: Wong
authors.forename: Babak
authors.initials: B
authors.lastname: Javid
authors.forename: Balasubrahmanyam
authors.initials: B
authors.lastname: Addepalli
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: Michael Brad
authors.initials: MB
authors.lastname: Strader
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
authors.forename: Clifton E
authors.initials: CE
authors.lastname: Barry
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation / Q000235:genetics; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D017433:Protein Structure, Secondary; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D012336:RNA, Ribosomal, 16S; D008780:Methyltransferases